While there are crystals of actin and of myosin, the molecular details of the interaction of myosin with actin are not well understood. There is a biochemical or modeled structural evidence for interaction between several areas of myosin with actin including a myosin surface loop that begins with amino acid 391 and ends with amino acid 404 in the human nonmuscle myosin IIA sequence based upon homology with the chicken skeletal muscle myosin that was crystallized. This loop is postulated to make direct contact with actin based on the Holmes-Rayment-Milligan model for interaction with actin and myosin. In human cardiac myosin, it contains a residue (Arg-403) that is mutated to a Gln or Trp to cause hypertrophic cardiomyopathy (HCM). In Acanthamoeba and Dictyostelium myosin I's and members of the myosin VI class, one of the residues in this loop is invariably a serine or threonine. In the myosin I molecules, phosphorylation of this Thr is required for activity. In almost all other myosins, this residue is either a Glu or Asp, suggesting that a negative charge at this residue is important for activity. We have used site-directed mutagenesis coupled with baculovirus expression of an HMM-like fragment of human nonmuscle IIA. We mutated Arg-393 (equivalent to the HCM mutation in cardiac myosin) to a Gln and Asp-399 (equivalent to the regulatory phosphorylation/invariant negative charged residue) to a Lys. Both mutants and wild-type HMM were well regulated by phosphorylation of the 20 kDa regulatory light chain. The Arg393Gln mutation results in a 25- 50% increase in the Vmax of the actin-activated MgATPase. The mutation did not affect the rate of actin filament sliding in an in vitro motility assay. The Asp399Lys mutation decreased the Vmax by 8-10 fold and the rate of in vitro motility by a factor of two, supporting an important role for the negative charge at this location. Neither mutation had a significant effect on the amount of actin required for half-maximal MgATPase activity and both are well regulated by their 20 kD light chain phosphorylation. We are currently studying other mutations of this loop.